The mesolimbic dopamine system plays an important role in mediating the rewarding effects of drugs of abuse including ethanol. Ethanol enhances a variety of properties of dopaminergic neurons including firing rate, dopamine turnover, metabolism, and release. The dopamine transporter (DAT), which is essential in terminating the actions of released dopamine, has been identified recently as a site of action for the effects of ethanol. We will test the overall hypothesis that DAT plays a pivotal role in maintaining dopaminergic homeostasis and dynamic transporter trafficking is an integral part of this process. In addition, normal DAT trafficking represents a compensatory mechanism to remove increased synaptic dopamine in response to drugs of abuse such as ethanol. The Specific Aims are: 1) To investigate ethanol's actions on DAT-mediated dopamine uptake kinetics and trafficking in mesolimbic mouse brain regions, 2) To discover novel proteins that interact with, and functionally regulate DAT in mesolimbic mouse brain regions, and 3) To investigate ethanol's effects on DAT trafficking and protein-protein interactions in cells expressing wild type and ethanolinsensitive mutant transporters. The experiments outlined for each aim will identify novel mechanisms underlying ethanol's action on transporter function. Compared to studies addressing ethanol's effects on postsynaptic receptor function, relatively few studies have focused on presynaptic neurotransmitter transporters as potential sites of action. The significance of the proposed work is the possibility of elucidating a new mechanism by which ethanol can regulate the activity of dopaminergic reward pathways. In addition, effects of ethanol on protein interactions and trafficking defined by this paradigm may also apply to other signaling systems. There are several innovative aspects of this research. First, there are few studies of ethanol action at presynaptic sites in general, and almost no attention to DAT, despite its importance in regulating dopaminergic neurotransmission. Second, use of proteomics techniques is very limited in alcohol research and this proposal provides applications of these innovations to the field. Third, the DAT proteome has not been studied, and defining this important complex will be an innovative accomplishment which should have wide ranging significance for other areas of neurobiology and addiction research.